Biology subjects

Ding, D.

Publications and source records attributed to Ding, D..

At least 19 recordsLinked to original sources

bioRxiv2026

A colorectal cancer organoid-CAF co-culture chip for drug screening and personalized therapy

The tumor microenvironment (TME) plays a critical role in cancer progression and therapeutic response, with cancer-associated fibroblasts (CAFs) being a key stromal component. Conventional tumor organoid models lack TME elements, and existing co-culture systems have limitations in recapitulating dynamic, multidimensional interactions. Here, we developed a novel dynamic co-culture chip (BAC) to better model the TME and investigate CAF-tumor cell crosstalk. Using this platform, we established a non-contact co-culture model of patient-derived colorectal cancer cells and CAFs. The resulting model closely recapitulated the morphological and molecular characteristics of the original patient tumors. Compared with tumor organoids cultured alone, the co-culture system exhibited significantly higher resistance to the clinically common chemotherapeutics 5-fluorouracil and oxaliplatin. Moreover, the presence of CAFs promoted tumor recurrence. Notably, drug responses in the co-culture model showed superior concordance with clinical outcomes relative to both organoid-only and animal models. Transcriptomic profiling under different culture conditions provided further insights into the mechanisms driving CAF-mediated interactions. These findings demonstrate that the BAC-based tumor organoid-CAF co-culture model serves as a more accurate platform for predicting drug responses, investigating TME-dependent mechanisms, and guiding personalized cancer therapy.

Cell Biology↗

bioRxiv2026

A hybrid framework integrating structural machine learning and 3D liver-on-chip assay for drug-induced liver injury prediction

Drug-induced liver injury (DILI) remains a major cause of clinical attrition and postmarketing withdrawal, but structure only DILI predictors are difficult to compare because public benchmarks are vulnerable to compound overlap, scaffold similarity and shared label provenance. We present OakuloidTM, an open DILI prediction framework that pairs a leakage audited structure based model with an optional iBAC 3D primary human hepatocyte IC50/Cmax confirmation signal. The structural model integrates gradient boosted descriptor backbones, fingerprint random forests and LivTox proxy-DILI features through a logistic meta learner. Its evaluation is designed as part of the contribution: internal DILIrank, strict external TDC, scaffold disjoint TDC and independent Geci provenance checks are reported with released per compound predictions. Oakuloid reaches AUROC 0.811 on the strict external TDC benchmark and remains competitive under scaffold and fully clean TDC filtering. A channel attribution ablation shows that the external benchmark lead is driven by descriptor based gradient boosted trees rather than by DILIPredictor derived proxy features, reducing a potential circularity concern. The wet lab IC50/Cmax signal is largely orthogonal to structure and supports a confirmation mode that shifts the internal operating point toward higher specificity without claiming a universal AUROC gain. Oakuloid is released with code, model artifacts, calibration analysis, a 122 compound wet lab benchmark and a model card under the Apache License 2.0, supporting reproducible DILI screening and benchmark auditing.

biochemistry↗

bioRxiv2026

CellFluxV2: An Image Generative Foundation Model for Virtual Cell Modeling

Building a virtual cell that simulates cellular behavior in silico is a central goal of computational biology. We introduce CellFluxV2, an image-generative model that predicts how cell morphologies change in response to chemical and genetic perturbations. A core innovation of CellFluxV2 is to learn distribution-level transformations from unperturbed to perturbed cells within the same experimental batch using flow matching, enabling it to disentangle true perturbation effects from confounding batch effects. Incorporating three methodological advances, CellFluxV2 achieves up to a 77% improvement in image fidelity over diffusion- and GAN-based baselines, while maintaining biological fidelity comparable to ground-truth images. Scaling up CellFluxV2, we establish the first scaling laws in image-based virtual cell modeling, demonstrating that performance improves consistently with both dataset size and model capacity. Furthermore, the scaled-up model generalizes well to out-of-distribution perturbations and exhibits two novel capabilities: batch-effect correction and cell-state interpolation. Together, these results position CellFluxV2 as a powerful foundation model advancing the vision of a virtual cell, unlocking novel opportunities for in silico drug screening.

bioinformatics↗

bioRxiv2026

Architecting cis-regulation to quantitatively tune gene expression in cereals

Precise modulation of gene expression via cis-regulatory editing holds promise for non-transgenic crop improvement, but the sequence-to-function relationships that govern plant promoter activity remain poorly understood. Here, we develop a massively parallel reporter assay (MPRA) in Sorghum bicolor to systematically measure the effects of >30,000 CRISPR-like mutations-deletions, substitutions, and motif insertions-across entire native promoters and 5' untranslated regions (UTRs) of three photosynthesis genes: PsbS, Raf1, and SBPase. We find that gene expression is most tunable within a [~]500 base pair core promoter region, where mutational effects are reproducible across biological replicates and predictive of protein output. Within these regions, we identify compact deletions and motif insertions that strongly increase protein production (>30-fold relative to wild type), exceeding the performance of transgenic enhancer elements. Mutation-effect relationships are gene-specific, highlighting the need for tailored regulatory maps. Our results establish a high-throughput strategy for cis-regulatory fine-mapping that enables crop improvements via minimal, precise, and non-transgenic gene edits.

synthetic biology↗

bioRxiv2025

Residual blinking-driven channel alignment for multicolor single-molecule localization microscopy

Given the nanometer-scale resolution of single-molecule localization microscopy (SMLM), sequential multicolor SMLM is inherently challenged by inter-channel misalignment arising from unpredictable sample drift during channel switching. Traditional fiducial-based strategies are limited by added complexity in sample preparation. Here, we report a residual blinking-driven method for channel alignment in multicolor SMLM, termed ReBling alignment. By exploiting residual blinking events under dual-wavelength excitation, our method extracts shared localizations as intrinsic registration anchors and performs accurate cross-correlation-based alignment. We validated ReBling alignment across biological systems featuring distinct spatial patterns, including continuous patterned structures (mitochondria-microtubule co-imaging, dual-labeled microtubules), and discrete point-cluster assemblies (dual-color nuclear pore complex components), demonstrating its broad applicability and reliability. These results establish ReBling alignment as a practical, versatile, fiducial-free solution for accurate channel registration in sequential multicolor SMLM, enabling more precise and reliable characterization of complex cellular architecture and molecular interactions.

cell biology↗

bioRxiv2025

Spatiotemporal control of PIWI compartmentalization by mitochondrial scaffolds defines pachytene piRNA pathway organization

Pachytene piRNAs are the least understood class of piRNAs in the mammalian male germ line. During meiosis, their biogenesis occurs near mitochondrial outer membrane in germ granules known as intermitochondrial cement (IMC). However, how mitochondrial factors regulate the trafficking of PIWI proteins into and out of the IMC remain poorly understood. Here we show that the cytoplasmic PIWI proteins MILI and MIWI are recruited for pachytene piRNA biogenesis via distinct mitochondrial membrane proteins. Loss of the mitochondrial scaffold protein ASZ1 during meiosis in mice disrupts multiple downstream biogenesis steps, leading to misregulation of MILI and MIWI, failure of IMC formation, and a near-complete loss of mature pachytene piRNAs. Strikingly, despite the drastic depletion of pachytene piRNAs, LINE1 transposon silencing remains unaffected. We identify three classes of pachytene piRNA pathway components that coordinate piRNA production and compartmentalization. Our findings reveal that chromatoid body precursors serve as a central hub for the accumulation of pachytene PIWI-piRNA complexes, thus establishing a connection between IMC-based biogenesis and downstream piRNA function.

developmental biology↗

bioRxiv2025

Pan-cancer analysis reveals that tumor microbiomes generate specific epitopes through transcriptional reprogramming

The therapeutic promise of tumor microbial peptides faces a critical safety paradox: tumors and peritumor tissues harbor compositionally similar microbiomes, yet safe immunotherapy demands tumor-specific targeting. We performed comprehensive transcriptomic analysis of microbial communities from 1,868 tumor and peritumor samples across 11 cancer types. Although 88.5% of microbial species are present in both tissue types, tumor and peritumor tissues exhibit profound transcriptional divergence: only 34.1% of microbial genes are expressed in both tissue types, and merely 18.2% of predicted HLA-binding peptides overlap. This transcriptional divergence represents microbial transcriptional reprogramming (MTR) in tumor tissues, through which microbes activate distinct genomic regions while adapting to tumor microenvironmental pressures, thereby generating tumor-specific peptide repertoires. Using Microbial Transcriptional Reprogramming Index (MiTRI) to quantify MTR, we found that higher MiTRI values correlate with expanded immunogenic peptide repertoires. Mass spectrometry-based immunopeptidomics confirmed that 25% of naturally presented microbial peptides originate from MTR regions. Crucially, in microsatellite-stable colorectal cancer, neo-adjuvant therapy responders exhibited significantly higher MiTRI values. T cell receptors (TCRs) recognized MTR-derived epitopes at 2.10-fold higher rates than genome-wide epitopes, accounting for 79.1% of clonally expanded TCRs. These findings establish MTR as the principle that ensures the tumor-specificity and safety of micorbiome-directed immunotherapy.

cancer biology↗

bioRxiv2025

Programmable Aptamer-Embedded Circular RNAs for Targeted Antigen-Presenting Cells Immunotherapy

The targeted delivery of mRNA to specific cell types in vivo remains a major unmet need in mRNA-based therapeutics. Conventional strategies rely on nanocarriers engineered with various modifications. However, these exogenous materials could cause adverse reactions, and their complex manufacturing processes often hinder clinical application. By leveraging the innate programmability of RNA sequences, we herein report a carrier-free, scalable, and programmable aptamer-embedded circular RNAs (Apt-circRNAs) design that confers targeting capability through the strategic incorporation of different aptamers at varying densities, while retaining circular RNA stability and protein production capability. Experimental results show that antigen-loaded Apt-circRNAs enable targeted delivery to antigen-presenting cells (APCs), drive endogenous antigen expression, activate antigen-specific T cells, and elicit potent immune responses that mediate clearance of both early-and late-stage tumors in mouse models. In humanized models of colorectal cancer, the combination of Apt-circRNA and PD-1 blockade produced synergistic antitumor effects and significantly reshaped the tumor microenvironment. In a first-in-human (FIH) clinical trial, Apt-circRNA vaccine encoding KRAS G12D/G12V neoantigens exhibited a favorable safety profile and induced robust immune activation, T cell mobilization, and neoantigen-specific immune responses. With this study, we have established a new paradigm in carrier-free, APC-targeted RNA vaccine design, offering a safe and effective platform for next-generation cancer immunotherapy.

bioengineering↗

bioRxiv2025

MGMG: Cell Morphology-Guided Molecule Generation for Drug Discovery

Designing novel molecules with desired bioactivity remains a fundamental challenge in drug discovery. Most molecular design methods follow target-based drug discovery paradigms that rely on well-defined drug targets, thereby limiting their applicability to diseases lacking known targets or reference compounds. Here we introduce Morphology-Guided Molecule Generation (MGMG), a phenotypic drug discovery-oriented approach that integrates cellular morphological profiles from compound treatments with molecular textual descriptions without requiring molecular target information. Cell morphology offers the guidance on desired bioactivity-relevant phenotypic effects, while textual descriptions provide direct and interpretable cues about molecular structure. Leveraging complementary structural and bioactivity context, MGMG significantly enhances molecule generation performance, especially in scenarios where textual descriptions are under-informative or morphological signals are weak. MGMG can also be applied to genetic perturbations, enabling activator design from gene overexpression morphology without requiring knowledge of reference compound structure. In addition, in silico docking demonstrates that MGMG-generated molecules, despite lacking target information, exhibit binding affinities comparable to reference compounds, preserving key interactions while introducing structural diversity. Overall, MGMG jointly utilizes morphological and textual description inputs to guide molecule generation, enabling diverse, bioactivity-aware compound design in a target-agnostic fashion.

bioinformatics↗

bioRxiv2025

A Geometrical Optics Model of Individual Ommatidia in Compound Eyes and a Hypothesis for Monocular Stereopsis

This study proposes a novel geometrical optics-based visual model suggesting that individual ommatidia in insect compound eyes may possess intrinsic three-dimensional imaging capabilities and enable monocular stereoscopic perception. Building upon the authors previous hypothesis that certain vertebrate retinal cell nuclei act as microlenses, the optical model is extended in this work to the arthropod compound eye system. By conceptualizing the cornea, crystalline cones, and layered rhabdomeric nuclei as components of a "multi-angle miniature real-image telescope," we construct a three-tiered imaging system composed of objectives, relay conjugate lenses, and microlenses. The model predicts that an ommatidium can actively perceive spatial depth and object distance by adjusting the focal length of crystalline cones and the distance between focused planes, thus achieving stereoscopic imaging with a single eye. Simulations based on representative species such as Drosophila melanogaster, Viggiani parasitic wasps, and fireflies (Lampyris) support the feasibility of this model in distance perception across scales ranging from microns to meters. The model breaks away from the "one ommatidium--one image point" paradigm and offers a new theoretical architecture for understanding insect stereopsis. It also presents a promising framework for bioinspired 3D imaging systems and optical designs with multi-focal capabilities.

biophysics↗

bioRxiv2025

Temporal and spatial composition of the tumor microenvironment predicts response to immune checkpoint inhibition

Immune checkpoint inhibition (ICI) has fundamentally changed cancer treatment. However, only a minority of patients with metastatic triple negative breast cancer (TNBC) benefit from ICI, and the determinants of response remain largely unknown. To better understand the factors influencing patient outcome, we assembled a longitudinal cohort with tissue from multiple timepoints, including primary tumor, pre-treatment metastatic tumor, and on-treatment metastatic tumor from 117 patients treated with ICI (nivolumab) in the phase II TONIC trial. We used highly multiplexed imaging to quantify the subcellular localization of 37 proteins in each tumor. To extract meaningful information from the imaging data, we developed SpaceCat, a computational pipeline that quantifies features from imaging data such as cell density, cell diversity, spatial structure, and functional marker expression. We applied SpaceCat to 678 images from 294 tumors, generating more than 800 distinct features per tumor. Spatial features were more predictive of patient outcome, including features like the degree of mixing between cancer and immune cells, the diversity of the neighboring immune cells surrounding cancer cells, and the degree of T cell infiltration at the tumor border. Non-spatial features, including the ratio between T cell subsets and cancer cells and PD-L1 levels on myeloid cells, were also associated with patient outcome. Surprisingly, we did not identify robust predictors of response in the primary tumors. In contrast, the metastatic tumors had numerous features which predicted response. Some of these features, such as the cellular diversity at the tumor border, were shared across timepoints, but many of the features, such as T cell infiltration at the tumor border, were predictive of response at only a single timepoint. We trained multivariate models on all of the features in the dataset, finding that we could accurately predict patient outcome from the pre-treatment metastatic tumors, with improved performance using the on-treatment tumors. We validated our findings in matched bulk RNA-seq data, finding the most informative features from the on-treatment samples. Our study highlights the importance of profiling sequential tumor biopsies to understand the evolution of the tumor microenvironment, elucidating the temporal and spatial dynamics underlying patient responses and underscoring the need for further research on the prognostic role of metastatic tissue and its utility in stratifying patients for ICI.

cancer biology↗

bioRxiv2025

A Geometric Optical Analysis of Human Retinal Cells and the Hypothesis of Monocular Stereoscopic Vision

We propose a geometric optical model that describes real-image formation via monocular vision in three dimensions. We only assume that the ganglion cell nuclei, bipolar cell nuclei, and photoreceptor cell nuclei in the human retina can be treated as microlenses and actively participate in imaging. In particular, we suggest that monocular vision operates via an array of high-magnification micro-real-image telescopes oriented in various azimuthal directions. The main elements of each telescope are the following. The first is the cornea and crystalline lens, which serve as objective lenses with tunable focal lengths. The second is a paired array of ganglion cell nuclei and bipolar cell nuclei, which act as a conjugate mirror. The third is an individual photoreceptor cell nuclei (cone or rod nuclei), which function as a micro-eyepiece. The fourth is the outer segment of each photoreceptor cell, which serves as a micro-object-distance sensor. We also hypothesize that six combinations of conjugate mirrors and micro-eyepiece arrays work within the human eye. Each combination is responsible for perceiving different ranges of object distances. We further hypothesize that the process by which one human eye perceives object distances involves two stages: from near to finite distances, and from finite distances to infinite distances. In different stages, the distance between different optical elements is tuned so that the object distance can be computed a specific formula when the photocurrent at the retina photoreceptors outer segment layer vanishes. Lastly, we conduct computational simulations. We find that there are two possible processes. By tuning the distances between the elements of our optical model, the eye can perceive object distances as close as 25 cm to dozens of meters in one process and dozens of meters to kilometers in the other process, depending on the sensitivity of the eye to small changes in focal plane spacing.

physiology↗

bioRxiv2024

H2B.W2, a Spermatocytes-specific Histone Variant, disrupts nucleosome stability and reduces chromatin compaction

Spermatogenesis is a highly regulated process that requires precise chromatin remodeling, which includes the incorporation of testis-specific histone variants. While several of these variants have been characterized, the role of H2B.W2, a member of the H2BW family, remains largely unclear. Here, we showed that H2B.W2 expression occurs mainly in spermatocytes, slightly later than its paralog H2B.W1. Cryo-electron microscopy (cryo-EM) analysis of H2B.W2-containing nucleosomes reveals a more relaxed conformation compared to canonical nucleosomes caused by weakened interactions between the outer DNA turn and the histone core. We pinpointed the N-terminal tail and 2 helix of H2B.W2, specifically residues D85 and Q101, as critical for nucleosome destabilization. Furthermore, we identify G73 within the L1 loop as a key residue involved in disrupting higher-order chromatin structure. Our findings suggest that H2B.W2-mediated nucleosome and chromatin destabilization may play a role in regulating gene expression during spermatogenesis, with potential implications for sperm development and function.

biochemistry↗

bioRxiv2024

Structure of the human taurine transporter TauT reveals substrate recognition and mechanisms of inhibition

Taurine, a sulfur-containing amino acid, plays a crucial role in human health due to its antioxidant, anti-inflammatory, osmoregulatory properties 1,2. Taurine levels are primarily regulated by the sodium-/chloride-dependent taurine transporter (TauT) 3. Inhibitors of TauT have potential in the treatment of certain diseases, including neurological disorders and cancer 4,5. Here, we present five structures of TauT: in the substrate-free apo state, in complex with taurine and in complex with taurine-mimetic inhibitors {beta}-alanine, GABA and guanidinoethyl sulfonate (GES), each with varying length of linear structure, in both inward-facing and occluded conformations. The taurine-, {beta}-alanine- and GABA-bound hTauT structures, in the presence of NaCl, adopt an occluded conformation, with ligands binding in the central pocket. In the presence of KCl, GES-bound hTauT adopts an inward-facing conformation, with two molecules positioned along the substrate translocation pathway with one into the deep central cavity and the other precluded conformational change from inward-facing to occluded state. Combined with function analysis, our structures provided insights into the overall architecture, substrate coordination and inhibitor recognition mechanisms of TauT.

biophysics↗

bioRxiv2024

TRPC3 suppression ameliorates synaptic dysfunctions and memory deficits in Alzheimer's disease

Transient receptor potential canonical (TRPC) channels are widely expressed in the brain; however, their precise roles in neurodegeneration, such as Alzheimers disease (AD) remain elusive. Bioinformatic analysis of the published single-cell RNA-seq data collected from AD patient cohorts indicates that the Trpc3 gene is uniquely upregulated in excitatory neurons. TRPC3 expression is also upregulated in post-mortem AD brains, and in both acute and chronic mouse models of AD. Functional screening of TRPC3 antagonists resulted in a lead inhibitor JW-65, which completely rescued A{beta}-induced neurotoxicity, impaired synaptic plasticity (e.g., LTP), and learning memory in acute and chronic experimental AD models. In cultured rat hippocampal neurons, we found that treatment with soluble {beta}-amyloid oligomers (A{beta}Os) induces rapid and sustained upregulation of the TRPC3 expression selectively in excitatory neurons. This aberrantly upregulated TRPC3 contributes to A{beta}Os-induced Ca2+ overload through the calcium entry and store-release mechanisms. The neuroprotective action of JW-65 is primarily mediated via restoring A{beta}Os-impaired Ca2+/calmodulin-mediated signaling pathways, including calmodulin kinases CaMKII/IV and calcineurin (CaN). The synaptic protective mechanism via TRPC3 inhibition was further supported by hippocampal RNA-seq data from the symptomatic 5xFAD mice after chronic treatment with JW-65. Overall, these findings not only validate TRPC3 as a novel therapeutic target for treating synaptic dysfunction of AD but most importantly, disclose a distinct role of upregulated TRPC3 in AD pathogenesis in mediating Ca2+ dyshomeostasis.

neuroscience↗

bioRxiv2024

Diaminopurine in Nonenzymatic RNA Template Copying

In the primordial RNA World, before the advent of ribozymes, nonenzymatic template copying would have been essential for the transmission of genetic information. However, the products of chemical copying with the canonical nucleotides are heavily biased towards the incorporation of G and C. Diaminopurine (D) can form a D:U base pair that is almost as strong as a G:C base pair. We therefore asked whether replacing A with D might lead to more efficient and less biased nonenzymatic template copying. As expected, primer extension substrates containing D bind to U in the template more tightly than substrates containing A. However, primer extension with D exhibited elevated reaction rates on a C template, leading to concerns about fidelity. To investigate the nature of the D:C mismatch, we solved the crystal structure of RNA duplexes containing D:C mismatches, and showed that D can form a wobble-type base pair with C. We then asked whether competition with G would decrease mismatched primer extension. We performed nonenzymatic primer extension with all four activated nucleotides on randomized RNA templates containing all four letters, and used deep sequencing to analyze the products. We found that the DUCG genetic system exhibited a more even product distribution and a lower mismatch frequency than the canonical AUCG system. Furthermore, primer extension is greatly reduced following all mismatches, including the D:C mismatch. Our study suggests that diaminopurine deserves further attention for its possible role in the RNA World, and as a potentially useful component of artificial nonenzymatic RNA replication systems.

biochemistry↗

bioRxiv2023

PNLDC1 catalysis and postnatal germline function are required for piRNA trimming, LINE1 silencing, and spermatogenesis in mice

PIWI-interacting RNAs (piRNAs) play critical and conserved roles in transposon silencing and gene regulation in the animal germline. Two distinct piRNA populations are present during mouse spermatogenesis: pre-pachytene piRNAs in fetal/neonatal testes and pachytene piRNAs in adult testes. PNLDC1 is required for both pre-pachytene piRNA and pachytene piRNA 3 end maturation in multiple species. However, whether PNLDC1 is the bona fide piRNA trimmer and the physiological role of 3 trimming of two distinct piRNA populations in spermatogenesis remain unclear. Here, by inactivating Pnldc1 exonuclease activity in vitro and in mice, we reveal that PNLDC1 trimmer activity is required for both pre-pachytene piRNA and pachytene piRNA 3 end trimming and male fertility. Furthermore, conditional inactivation of Pnldc1 in postnatal germ cells causes LINE1 transposon de-repression and spermatogenic arrest in mice. This indicates that pachytene piRNA trimming, but not pre-pachytene piRNA trimming, is essential for mouse germ cell development and transposon silencing. Our findings highlight the potential of inhibiting germline piRNA trimmer activity as a potential means for male contraception.

developmental biology↗

bioRxiv2023

A conserved N-terminal motif of CUL3 mediates assembly and licenses E3 ligase activation of CRL3KLHL22

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through E3-E3 dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that mediates the dimerization assembly and licenses the activation of CRL3KLHL22. Deletion of the CUL3 N-terminal motif leads to non-stochiometric assembly and impairs E3 activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric E3-E3 superassembly generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate a CUL3 N-terminal motif-dependent E3-E3 superassembly mechanism and provide insights into the assembly and activation of CRL3s.

molecular biology↗